Project Summary: In 2017 head and neck squamous cell carcinoma (HNSCC) will result in 49,700 new cases in the United States and an estimated 500,000 cases worldwide. Radiation is standard of care in many cases. Unfortunately, a significant percentage of HNSCCs are also hypoxic, making them markedly more resistant to radiotherapy than healthy tissue. This resistance has been shown to impair treatment response associated with a higher risk of recurrence and metastasis. While approaches using systemic oxygen (O2) delivery have largely stalled, our group has shown substantial improvements in tumor control and survival in breast cancer models using an ultrasound sensitive, surfactant-based O2 microbubble platform to overcome tumor hypoxia immediately prior to radiotherapy. After intravenous injection, ultrasound can be used to noninvasively rupture the bubbles, triggering a localized intratumoral release of O2. While promising, clinical translation of this platform is not yet feasible as oxygenation is limited to 2-3 minutes. Our hypothesis is we can overcome these limitations using a combination therapy of oxygen delivery with lonidamine and metformin (two pharmaceutical agents that selectively target the metabolic pathway in tumors primarily by mitochondrial respiration). We expect the addition of these agents will prolong oxygenation while also improving systemic anti-tumor immune responses (via the abscopal effect) and extend therapeutic effects beyond the primary tumor to metastatic disease. The proposed work is logically divided into three logical aims each of which is supported by sufficient preliminary data to not require inter-dependence across any aims. In Aim 1 we will modify our previous design to also encapsulate lonidamine (whose bioavailability has limited clinical translation). Microbubbles will be optimized to maximize stability and payload before being acoustically characterized and validated in hypoxic cell culture. Aim 2 will comprise of in vivo O2 monitoring experiments with and without oral metformin to compare previous O2 microbubble designs and controls in order to define optimal radiotherapy treatment parameters and clinical feasibility. Biodistribution studies will also be performed to evaluate local delivery of lonidamine. Finally, in Aim 3, the platform and selected timing and acoustic parameters will be validated in vivo in tumor-bearing mice using established and well-characterized HNSCC models. We will determine the ability of O2 microbubbles to effectively sensitize tumors to radiation (evaluated by tumor growth kinetics and effects on survival), while also exploring the platform's effects on increased immune response in the tumor microenvironment in immunocompetent mice. At the conclusion of this project, we will have developed and validated a minimally invasive and clinically translatable method to overcome tumor hypoxia prior to radiotherapy. We expect that this platform will improve patient outcomes by improving responses to radiotherapy and potentially reduce the prevalence of recurrence and metastasis of HNSCC after treatment.